CN116171308A - Fluorescent dyes - Google Patents

Abstract

Automated oligonucleotide synthesis compatible fluorescent dye phosphoramidite compounds, solid supports and labeled polynucleotides incorporating the compounds are provided. The compounds allow for the ubiquitous incorporation of fluorescent labels anywhere in a polynucleotide.

Description

Fluorescent dye

Cross Reference to Related Applications

The present application claims priority from U.S. application No. 63/052,862 filed on 7/16/2020, which is expressly incorporated herein by reference in its entirety.

Background

Fluorescent dyes are one of the most commonly used labels for modifying oligonucleotides, as they provide sensitive detection in a variety of applications ranging from PCR to sequencing. Preparation of fluorescent dye-labeled polynucleotides is typically accomplished by post-synthesis conjugation, for example, by reacting an activated dye intermediate with an amino derivative of the polynucleotide. This method suffers from certain drawbacks including low conjugation yields and the need for additional purification of the conjugated product. Incorporation of fluorescent dyes into synthetic polynucleotides via automated phosphoramidite synthesis provides a more convenient method. However, few phosphoramidite derivatives of fluorescent dyes are commercially available that allow for the addition of fluorescent dyes to polynucleotides as part of automated solid phase synthesis. In addition, there are few such derivatives that can allow incorporation of fluorescent dye moieties at any position in the synthesized polynucleotide.

Thus, there remains a need for fluorescent dyes that are compatible with automated phosphoramidite oligonucleotide synthesis conditions, can be incorporated at any position of a polynucleotide, and provide high endpoint fluorescent signals in PCR applications.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the present disclosure provides a compound represented by formula I, or a stereoisomer, salt, or tautomer thereof:

wherein:

x is H, halogen or C ₁ -C ₅ An alkyl group; r is R ¹ 、R ² 、R ³ And R is ⁴ Independently H or optionally substituted C ₁ -C ₆ An alkyl group; r is R ⁵ 、R ⁶ 、R ⁷ 、R ⁸ And R is ⁹ Independently H, halogen or optionally substituted C ₁ -C ₆ An alkyl group; l (L) ¹ Is optionally substituted C ₂ -C ₁₀ Alkylene or optionally substituted C ₂ -C ₅₀ A heteroalkylene group; l (L) ² And L ³ Independently optionally substituted C ₂ -C ₁₀ Alkylene or optionally substituted C ₂ -C ₃₀ A heteroalkylene group; q (Q) ¹ Is a hydroxyl protecting group; z isCH. N, NHC (O) N or OC (O) N; y is OH, OP (OCH) ₂ CH ₂ CN)NR ¹⁰ R ¹¹ Or a solid support; r is as follows ¹⁰ And R is ¹¹ Independently optionally substituted C ₁ -C ₆ An alkyl group.

In some embodiments, X is Cl, br, or F. In some embodiments, X is Cl. In some embodiments, X is F. In some embodiments, X is optionally substituted methyl or optionally substituted ethyl. In some embodiments, X is o-MeC ₆ H ₄ CH ₂ 。

In some embodiments, the solid support is a controlled pore glass or polystyrene.

In some embodiments, the compound is a compound of formula (IA):

in some embodiments, R ¹ Is H. In some embodiments, R ² Is methyl. In some embodiments, R ³ Is methyl. In some embodiments, R ⁴ Is methyl. In some embodiments, the compound is a compound of formula (IB):

in some embodiments, L ¹ Is C ₂ -C ₆ Alkylene or- (CH) ₂ CH ₂ O) _m CH ₂ CH ₂ -wherein m is an integer from 1 to 10. In some embodiments, L ¹ Is C ₂ An alkylene group. In some embodiments, Z is OC (O) N.

In some embodiments, the compound is a compound of formula (IC) or a stereoisomer, salt, or tautomer thereof:

wherein m is an integer of 1 to 10.

In some embodiments, the compound is a compound of formula (ID) or a stereoisomer, salt, or tautomer thereof:

wherein W is NH or O, and L ⁴ Is optionally substituted C ₂ -C ₁₀ Alkylene or optionally substituted C ₂ -C ₅₀ A heteroalkylene group.

In some embodiments, Q ¹ Is trimethylsilyl, TBDMS, acetyl, dimethoxytrityl or trityl. In some embodiments, R ¹⁰ And R is ¹¹ Is isopropyl. In some embodiments, R ⁵ Is methyl. In some embodiments, R ⁷ Is methyl. In some embodiments, R ⁸ Is methyl. In some embodiments, R ⁶ And R is ⁹ Is H.

In some embodiments, the compound is a compound of formula (IE) or a stereoisomer, salt, or tautomer thereof:

in another aspect, the disclosure provides a labeled polynucleotide comprising a residue of a compound of the disclosure. In some embodiments, the labeled polynucleotides are prepared using automated phosphoramidite synthesis. In some embodiments, the labeled polynucleotide further comprises a fluorescence quencher. In some embodiments, the labeled polynucleotide further comprises a BHQ-type fluorescence quencher. In some embodiments, the labeled polynucleotide is attached to a solid support. In some embodiments, the solid support is a controlled pore glass bead, polystyrene bead, magnetic bead, or microplate.

In another aspect, the present disclosure provides a method for preparing a labeled conjugate of a ligand comprising contacting the ligand with a compound of any one of claims 1 to 22 in a suitable solvent under conditions sufficient to covalently attach the compound to the ligand, thereby forming a labeled conjugate, wherein Y is OP (OCH) ₂ CH ₂ CN)NR ¹⁰ R ¹¹ And R is ¹⁰ And R is ¹¹ Independently optionally substituted C ₁ -C ₆ An alkyl group.

In some embodiments, the ligand is a polynucleotide or a solid support. In some embodiments, the ligand is a polynucleotide. In some embodiments, the conditions sufficient to covalently link the compound to the ligand are automated phosphoramidite oligonucleotide synthesis conditions.

In another aspect, the present disclosure provides kits, such as PCR diagnostic kits, comprising the labeled polynucleotides disclosed herein.

Detailed description of the preferred embodiments

Provided herein are fluorescent dye derivatives, such as phosphoramidites, which can be incorporated into oligonucleotides via standard automated oligonucleotide synthesis. Solid supports modified with fluorescent dyes and polynucleotides comprising fluorescent dyes are also provided.

In this specification and the appended claims, the words "comprise" and "include" and variations thereof, such as "comprises", "comprising", "including" and "including" are intended to convey that other elements or integers not specifically recited may be included where the context permits. As used herein, the term "consisting of" is intended to mean including and limited to any content following the phrase "consisting of". Thus, the phrase "consisting of" means that the listed elements are essential or mandatory and that no other elements are present. The term "consisting essentially of means that the composition, method, or structure may include additional ingredients, steps, and/or portions, provided that the additional ingredients, steps, and/or portions do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

The terms "a" and "an" and "the" and similar referents are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each group member may be cited and claimed alone or in any combination with other members of the group or other elements found herein. It is contemplated that one or more members of a group may be included in or deleted from the group for convenience and/or patentability reasons. When any such inclusion or deletion occurs, the specification is considered herein to contain the modified group, thereby satisfying the written description of all markush groups used in the appended claims.

Unless otherwise indicated, nucleic acids or oligonucleotides are written in the 5 'to 3' direction from left to right.

As used herein, the term "amplification" refers to any manner of generating at least a portion of the sequence of at least one target nucleic acid or its sequence complement, typically in a template-dependent manner, including, but not limited to, broad techniques for linear or exponential amplification of nucleic acid sequences. Non-limiting exemplary amplification methods include Polymerase Chain Reaction (PCR), reverse transcriptase PCR, real-time PCR, nested PCR, multiplex PCR, quantitative PCR (Q-PCR), nucleic Acid Sequence Based Amplification (NASBA), transcription Mediated Amplification (TMA), ligase Chain Reaction (LCR), rolling Circle Amplification (RCA), strand Displacement Amplification (SDA), ligase Detection Reaction (LDR), multiplex ligation dependent probe amplification (MLPA), Q-replicase amplification after ligation, primer extension, strand Displacement Amplification (SDA), hyperbranched strand displacement amplification, multiplex Displacement Amplification (MDA), nucleic Acid Strand Based Amplification (NASBA), two-step multiplex amplification, digital amplification, and the like. Descriptions of such techniques are found in Ausubel et al; PCR primer: laboratory manual (A Laboratory Manual), diffenbach edit, cold Spring Harbor Press (1995); electronic protocol (The Electronic Protocol Book), chang Bioscience (2002); nucleic acid protocol manual (The Nucleic Acid Protocols Handbook), r.rapley editions, humana Press, totowa, n.j. (2002); and Innis et al, PCR Protocols (PCR Protocols): methods and application guidelines (A Guide to Methods and Applications), academic Press (1990).

As used herein, the term "base" refers to a nitrogen-containing heterocyclic moiety capable of forming hydrogen bonds, such as Watson-Crick (Watson-Crick) type hydrogen bonds, with a complementary nucleotide base or nucleotide base analog, such as a purine, 7. Deazapurine, or pyrimidine. Typical bases are naturally occurring bases adenine, cytosine, guanine, thymine and uracil. Bases also include analogs of naturally occurring bases such as deazaadenine, 7-deaza-8-azaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, inosine, gourmet, nitropyrrole, nitroindole, 2-aminopurine, 2, 6-diaminopurine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-chloro-6-aminopurine, xanthine, hypoxanthine, and the like.

As used herein, the term "complementary" refers to the ability of polynucleotide sequences to hybridize to and from base pairs with each other. Base pairs are typically formed by hydrogen bonding between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands may base pair in a Watson-Crick manner (e.g., A through T, A through U, C through G) or in any other manner that allows duplex formation. The percent "complementarity" of a probe sequence to a target sequence is the percent "identity" of the probe sequence to the target sequence or complement of the target sequence. In determining the degree of "complementarity" between a probe and a target sequence, the degree of "complementarity" is expressed as the percent identity between the probe sequence and the sequence of the target sequence that best matches it or the complement of the sequence of the target sequence. Exemplary probes are polynucleotides described herein.

As used herein, the term "duplex" refers to a double-stranded hybridization complex formed by annealing (hybridizing) complementary (or partially complementary) single-stranded polynucleotides, such as DNA, RNA, LNA or Peptide Nucleic Acids (PNAs).

As used herein, "fluorescence quenching" refers to any process that reduces the fluorescence intensity of a fluorescent sample, i.e., a fluorescent polynucleotide probe. Multiple molecular interactions can lead to quenching. Non-limiting examples include excited state reactions, molecular rearrangements, energy transfer, ground state complex formation, and collision quenching.

As used herein, "halogen" refers to F, cl, br or I.

The terms "hybridization" and "hybridization" are used herein to refer to "specific hybridization," which in some embodiments is the preferential binding, duplexing, or annealing of a nucleic acid molecule to a particular nucleotide sequence under stringent conditions. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target sequence, and to a lesser extent to other sequences, or not to other sequences at all. "stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization are sequence-dependent and differ under different environmental parameters. A detailed guide to nucleic acid hybridization can be found, for example, in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I, ch.2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, NY. The degree of hybridization, also known as the hybridization intensity, of a polynucleotide to a target sequence is determined by methods well known in the art. One preferred method is to determine the T of a given hybridization duplex _m . This can be achieved by subjecting the duplex formed in solution to progressively higher temperatures and monitoring the denaturation of the duplex, for example by absorption of ultraviolet light, which increases with the de-stacking of base pairs accompanying denaturation. T (T) _m Generally defined as the temperature at which half of the DNA strand is in the single stranded (ssDNA) state. T (T) _m Depending on various parametersSuch as the length of the hybridized complementary strand sequences, their specific nucleotide sequences, base composition, base modification, and the concentration of the complementary strand.

As used herein, the terms "label" and "detectable label" are used interchangeably and refer to a moiety that, when attached to a biomolecule, nucleoside, nucleotide or polynucleotide, allows such biomolecule, nucleoside, nucleotide or polynucleotide to be detected by suitable detection means. Exemplary labels include fluorophores, chromophores, radioisotopes, spin labels, enzyme labels, chemiluminescent labels, electrochemiluminescent compounds, magnetic labels, microspheres, colloidal metals, immunological labels, ligands, enzymes, and the like.

As used herein, the term "modified nucleotide base" or "modified base" refers to a base that does not have the structure of a naturally occurring base and is therefore non-naturally occurring. As used herein, the term "modified sugar" refers to a sugar or sugar analogue that does not have the structure of a naturally occurring sugar, such as ribose or deoxyribose sugar, and thus is non-naturally occurring.

As used herein, the term "naturally occurring" in the context of a nucleic acid molecule refers to an RNA or DNA molecule (single or double stranded) having a naturally occurring nucleotide sequence and comprising only naturally occurring components such as bases, sugars, nucleosides, and nucleotides.

As used herein, the term "nucleoside" refers to a molecule consisting of a nitrogenous base of the type mentioned herein that is bound to a sugar of the type mentioned herein, for example to ribose or deoxyribose sugar, via a β -glycosidic linkage. Examples of nucleosides include adenosine, cytidine, guanosine, thymidine, uridine, and inosine.

As used herein, the term "nucleotide" refers to a phosphate ester of a nucleoside, either as an independent monomer or as a subunit within a polynucleotide. Nucleotide monomers include, for example, nucleotide 5 '-monophosphate, nucleotide 5' -diphosphate, nucleotide 5 '-triphosphate, and nucleotide 3' -monophosphate. Nucleotide triphosphates are sometimes denoted "NTP", "dNTP" (2 ' -deoxypentose) or "ddNTP" (2 ',3' -dideoxypentose) to specifically identify structural features of ribose sugars. "nucleotide 5 '-triphosphate" refers to a nucleotide having a triphosphate group at the 5' position. The triphosphate group may include a sulfur substitution of one or more phosphate oxygen atoms, such as an alpha-thio nucleotide 5' -triphosphate. Nucleotide monophosphates, nucleotide diphosphate or nucleotide triphosphate can be used as substrates for nucleic acid processing enzymes that catalyze the modification of nucleic acids or nucleic acid intermediates.

As used herein, the term "oligonucleotide" broadly refers to a single strand consisting essentially or entirely of about 2 to about 300 naturally occurring or modified nucleotide monomer units, such as deoxyribose or ribose sugar rings substituted with A, C, G, T or U bases, and which are linked by a conventional phosphate backbone moiety. More specifically, the term refers to single stranded deoxyribonucleotides within the above-mentioned size range. In some embodiments, the oligonucleotide may comprise one or more modified bases and/or sugars. In addition to the nucleotide monomer units, the oligonucleotides may incorporate one or more detectable labels and/or one or more reactive groups.

The term "plurality" as used herein means more than one.

As used herein, the term "polynucleotide" generally refers to an oligonucleotide comprising from about 10 to about 300 nucleotide monomer units. In addition to nucleotide monomer units, polynucleotides may incorporate one or more detectable labels and/or one or more reactive groups.

As used herein, the term "primer" refers to a polynucleotide or modified polynucleotide that is effective as a starting point for synthesis of a polynucleotide strand complementary to a target nucleic acid strand. For example, primers for PCR include forward and reverse primers, wherein the forward primer contains a sequence complementary to a region of a target nucleic acid strand and directs synthesis of the complementary strand. The reverse primer contains a sequence complementary to the opposite strand and directs synthesis of the opposite strand along the target nucleic acid strand.

As used herein, the term "probe" refers to a labeled oligonucleotide or labeled modified oligonucleotide that contains a sequence complementary to a region of a target nucleic acid sequence, allowing the probe to form a duplex with the target sequence and producing an indication of the region of the target sequenceA detectable signal is present. The detectable signal is generated directly or indirectly during or after hybridization. In certain applications, such as during primer extension in 5 '-nuclease PCR, the probe lacks an extendable 3' hydroxyl group to prevent polymerase mediated probe extension. In certain embodiments, the probe comprises

Probe, taqMan->

Probe, & lt/EN & gt>

Probes, molecular beacons (e.g., tyagi, sanjay&Kramer, fred (2012) Molecular Beacons in diagnostics, f1000 media reports, 4.10.10.3410/M4-10), and the like.

As used herein, the term "protecting group", "protecting group" or "protected form" refers to an unstable chemical modification of a functional group (e.g., hydroxyl group) intended to preserve its functionality and/or to obtain chemoselectivity in subsequent reactions. The protecting groups are removed from the final product by deprotection treatment (e.g., with acid).

As used herein, the term "solid support" refers to any insoluble material, including particles (e.g., beads), fibers, monoliths, membranes, filter materials, plastic strips, arrays, microplates, and the like. In some embodiments, the solid support is a solid support suitable for automated phosphoramidite oligonucleotide synthesis, such as polystyrene and Controlled Pore Glass (CPG).

In one aspect, provided herein are fluorescent dye compounds represented by formula I, or stereoisomers, salts, or tautomers thereof:

wherein:

x is H, halogen or optionally substituted C _1- C ₅ An alkyl group;

R ¹ 、R ² 、R ³ and R is ⁴ Independently H or optionally substituted C ₁ -C ₆ An alkyl group;

R ⁵ 、R ⁶ 、R ⁷ 、R ⁸ and R is ⁹ Independently H, halogen or optionally substituted C ₁ -C ₆ An alkyl group;

L ¹ is selected from optionally substituted C ₂ -C ₁₀ Alkylene or optionally substituted C ₂ -C ₅₀ A heteroalkylene linker group;

L ² and L ³ Independently selected from optionally substituted C ₂ -C ₁₀ Alkylene or optionally substituted C ₂ -C ₃₀ A heteroalkylene linker group;

Q ¹ protecting groups for hydroxyl groups, such as protecting groups compatible with automated oligonucleotide synthesis;

z is CH, N, NHC (O) N or OC (O) N;

y is OH, OP (OCH) ₂ CH ₂ CN)NR ¹⁰ R ¹¹ Or a solid support; and

R ¹⁰ and R is ¹¹ Independently optionally substituted C ₁ -C ₆ An alkyl group.

In some embodiments, formula I comprises a phosphoramidite group (i.e., Y is OP (OCH) ₂ CH ₂ CN)NR ¹⁰ R ¹¹ ) And hydroxyl groups protected with acid labile protecting groups such as trityl or dimethoxytrityl groups. In some embodiments, Q ¹ Is a dimethoxytrityl group. In some embodiments, R ¹⁰ And R is ¹¹ Is isopropyl.

In some embodiments of formula I, X is Cl, br or F. In some embodiments, X is Cl.

In some embodiments, the compound is a compound of formula (IA):

in some embodiments, R ¹ Is H. In some embodiments, R ² Is methyl. In some embodiments, R ³ Is methyl. In some embodiments, R ⁴ Is methyl. In some embodiments, R ¹ Is H, and R ² 、R ³ And R is ⁴ Each of which is methyl.

In some embodiments of formula (I), the compound is a compound of formula (IB):

in the formulae disclosed herein, the linker group L ¹ May comprise one or more heteroatoms selected from N, O, S, P and combinations thereof. In some embodiments, L ¹ Is PEG _2-10 A connector. In some embodiments, L ¹ Is C ₂ -C ₆ Alkylene or- (CH) ₂ CH ₂ O) _m CH ₂ CH ₂ -wherein m is an integer from 1 to 10. In other embodiments, L ¹ Is optionally substituted alkylene, such as-CH ₂ CH ₂ -. In some embodiments, L ¹ Is an alkylene group optionally substituted with a methyl group.

In some embodiments, Z is OC (O) N.

In some embodiments of formulas (I), (IA) and (IB), the compound is a compound of formula (IC):

wherein m is an integer of 1 to 10.

In some embodiments of formula (I), the compound is a compound of formula (ID):

wherein W is NH or O, and L ⁴ Is optionally substituted C ₂ -C ₁₀ Alkylene or optionally substituted C ₂ -C ₅₀ A heteroalkylene group.

In the formulae shown herein, Q ¹ Represents a hydroxyl protecting group. Examples of such protecting groups are known in the art (see, e.g., peter g.m.wuts, greene's protective groups in organic synthesis (2006)). Suitable hydroxyl protecting groups include base labile groups and acid labile groups. In some embodiments, Q is a hydroxyl protecting group compatible with the conditions of automated phosphoramidite oligonucleotide synthesis, such as a trityl or dimethoxytrityl group. In some embodiments, Q ¹ Is trimethylsilyl, TBDMS, acetyl, dimethoxytrityl or trityl.

In some embodiments, R ⁵ Methyl or halogen such as Cl or F. In some embodiments, R ⁷ Methyl or halogen such as Cl or F. In some embodiments, R ⁸ Methyl or halogen such as Cl or F. In some embodiments, R ⁷ Is H. In some embodiments, R ⁶ And R is ⁹ Is H.

In some embodiments, the compound is a compound of formula (IE) or a stereoisomer, salt, or tautomer thereof:

in some embodiments of formula (I), Y is a solid support such as a controlled pore glass or polystyrene. In some embodiments of formula (I), Y optionally comprises a linking group that links the controlled pore glass or polystyrene to the rest of the structure.

In another aspect, the present disclosure provides a compound of formula II, or a stereoisomer, salt, or tautomer thereof:

wherein X is H, halogen or optionally substituted C ₁ -C ₅ An alkyl group;

R ¹ 、R ² 、R ³ and R is ⁴ Independently H or optionally substituted C ₁ -C ₆ An alkyl group; and

R ⁵ 、R ⁶ 、R ⁷ 、R ⁸ and R is ⁹ Independently H, halogen or optionally substituted C ₁ -C ₆ An alkyl group.

In some embodiments of formula II, X is Cl, br or F. In some embodiments, X is Cl. In some embodiments, X is F.

In some embodiments of formula II, R ¹ Is H. In some embodiments, R ² Is methyl. In some embodiments, R ³ Is methyl. In some embodiments, R ⁴ Is methyl. In some embodiments, R ¹ Is H, and R ² 、R ³ And R is ⁴ Each of which is methyl.

As used herein, the terms "alkyl", "alkenyl" and "alkynyl" include straight, branched and cyclic monovalent hydrocarbon groups, as well as combinations of these, which when unsubstituted, contain only C and H. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl and the like. The total number of carbon atoms in each such group is sometimes described herein, and, for example, when the group may contain up to ten carbon atoms, it may be represented as 1-10C, C ₁ -C ₁₀ C-C10 or C1-10.

The terms "heteroalkyl", "heteroalkenyl" and "heteroalkynyl" as used herein refer to the corresponding hydrocarbon in which one or more chain carbon atoms have been replaced with a heteroatom. Exemplary heteroatoms include N, O, S and P. When a heteroatom is allowed to replace a carbon atom, for example, in a heteroalkyl group, although still written as, for example, C3-C10, the number describing the group represents the sum of the number of carbon atoms in the ring or chain and the number of such heteroatoms included as a replacement for the carbon atoms in the ring or chain as described.

Typically, alkyl, alkenyl and alkynyl substituents contain 1 to 10 carbon atoms (alkyl) or 2 to 10 carbon atoms (alkenyl or alkynyl). Preferably, they contain 1 to 8 carbon atoms (alkyl) or 2 to 8 carbon atoms (alkenyl or alkynyl). Sometimes they contain 1-6 carbon atoms (alkyl) or 2-6 carbon atoms (alkenyl or alkynyl). A single group may include more than one type of multiple bond, or more than one multiple bond; such groups are included in the definition of the term "alkenyl" when they contain at least one carbon-carbon double bond, and in the term "alkynyl" when they contain at least one carbon-carbon triple bond.

As used herein, the terms "alkylene", "alkenylene" and "alkynylene" include straight, branched, and cyclic divalent hydrocarbon groups and combinations thereof.

Alkyl, alkenyl and alkynyl groups may optionally be substituted to the extent that such substitution is chemically meaningful. Typical substituents include, but are not limited to, halogen (F, cl, br, I), =o, =n-CN, =n-OR, =nr, OR, NR ₂ 、SR、SO ₂ R、SO ₂ NR ₂ 、NRSO ₂ R、NRCONR ₂ 、NRC(O)OR、NRC(O)R、CN、C(O)OR、C(O)NR ₂ OC (O) R, C (O) R and NO ₂ Wherein each R is independently H, C ₁ -C ₈ Alkyl, C ₂ -C ₈ Heteroalkyl, C ₁ -C ₈ Acyl, C ₂ -C ₈ Heteroacyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Heteroalkenyl, C ₂ -C ₈ Alkynyl, C ₂ -C ₈ Heteroalkynyl, C ₆ -C ₁₀ Aryl or C ₅ -C ₁₀ Heteroaryl, and each R is optionally substituted with halo (F, cl, br, I), =o, =n-CN, =n-OR ',=nr', OR ', NR' ₂ 、SR′、SO ₂ R′、SO ₂ NR′ ₂ 、NR′SO ₂ R′、NR′CONR′ ₂ 、NR′C(O)OR′、NR′C(O)R′、CN、C(O)OR′、C(O)NR′ ₂ OC (O) R ', C (O) R' and NO ₂ Substitution wherein each R' is independently H, C ₁ -C ₈ Alkyl, C ₂ -C ₈ Heteroalkyl, C ₁ -C ₈ Acyl, C ₂ -C ₈ Heteroacyl, C ₆ -C ₁₀ Aryl, or C ₅ -C ₁₀ Heteroaryl groups. Alkyl, alkenyl and alkynyl groups may also be substituted with C ₁ -C ₈ Acyl, C ₂ -C ₈ Heteroacyl, C ₆ -C ₁₀ Aryl or C ₅ -C ₁₀ Heteroaryl substitution, each of which may be substituted with substituents appropriate to the particular group.

Although "alkyl" as used herein includes cycloalkyl and cycloalkylalkyl groups, the term "cycloalkyl" is used herein to describe carbocyclic non-aromatic groups attached via a ring carbon atom, and "cycloalkylalkyl" is used to describe carbocyclic non-aromatic groups attached to a molecule through an alkyl linker. Similarly, "heterocyclyl" is used to identify a non-aromatic ring group containing at least one heteroatom as a ring member and attached to the molecule via a ring atom, which may be C or N; and "heterocyclylalkyl" may be used to describe such a group attached to another molecule through an alkylene linker. As used herein, these terms also include rings containing one double bond or two double bonds, provided that the ring is not aromatic.

"aromatic" or "aryl" substituents or moieties refer to monocyclic or fused bicyclic moieties having well-known aromatic characteristics; examples include phenyl and naphthyl. Similarly, the terms "heteroaromatic" and "heteroaryl" refer to such single or fused bicyclic ring systems containing one or more heteroatoms as ring members. Suitable heteroatoms include N, O and S, their inclusion allows for aromaticity in 5-membered and 6-membered rings. Typical heteroaromatic systems include monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidinyl, pyrazinyl, thienyl, furyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl and imidazolyl, and fused bicyclic moieties formed by fusing one of these monocyclic groups with a benzene ring or any heteroaromatic monocyclic group to form a C8-C10 bicyclic group, such as indolyl, benzimidazolyl, indazolyl, benzotriazole, isoquinolyl, quinolinyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like. Any single or fused ring bicyclic ring system having an aromatic character in terms of the electron distribution throughout the ring system is included in this definition. It also includes bicyclic groups in which at least the ring directly attached to the remainder of the molecule has aromatic character. Typically, ring systems contain 5 to 12 ring member atoms. Preferably, monocyclic heteroaryl groups contain 5-6 ring members, and bicyclic heteroaryl groups contain 8-10 ring members.

The aryl and heteroaryl moieties may be substituted with various substituents including C ₁ -C ₈ Alkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₅ -C ₁₂ Aryl, C ₁ -C ₈ Acyl groups, as well as hybrid forms of these, each of which may themselves be further substituted; other substituents for the aryl and heteroaryl moieties include halogen (F, cl, br, I), OR, NR ₂ 、SR、SO ₂ R、SO ₂ NR ₂ 、NRSO ₂ R、NRCONR ₂ 、NRC(O)OR、NRC(O)R、CN、C(O)OR、C(O)NR ₂ OC (O) R, C (O) R and NO ₂ Wherein each R is independently H, C ₁ -C ₈ Alkyl, C ₂ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Heteroalkenyl, C ₂ -C ₈ Alkynyl, C ₂ -C ₈ Heteroalkynyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₇ -C ₁₂ Arylalkyl or C ₆ -C ₁₂ Heteroarylalkyl, and each R is optionally substituted as described above for the alkyl group. Substituents on aryl or heteroaryl groups may of course be further substituted with groups suitable for each type of such substituents or for each component of substituents described herein. Thus, for example, arylalkyl substituents can be substituted on the aryl moiety with pairs described hereinTypical substituents for aryl groups are substituted, and it may be further substituted on the alkyl moiety with substituents described herein that are typical or suitable for alkyl groups.

As used herein, "optionally substituted" means that the particular group described may have one or more hydrogen substituents substituted with non-hydrogen substituents. In some optionally substituted groups or moieties, all hydrogen substituents are substituted with non-hydrogen substituents such as C ₁ -C ₆ Alkyl, C ₂ -C ₆ Heteroalkyl, alkynyl, halo (F, cl, br, I), N ₃ 、OR、NR ₂ 、SR、SO ₂ R、SO ₂ NR ₂ 、NRSO ₂ R、NRCONR ₂ 、NRC(O)OR、NRC(O)R、CN、C(O)OR、C(O)NR ₂ OC (O) R, C (O) R, oxo and NO ₂ Wherein each R is independently H, C ₁ -C ₆ Alkyl or C ₂ -C ₆ A heteroalkyl group. When one optional substituent is linked via a double bond such as oxo or oxo (=o), the group occupies two available valences, so the total number of substituents that can be included decreases according to the number of available valences.

Salts, stereoisomers, and tautomers of the compounds disclosed herein are also within the scope of the disclosure. As used herein, "stereoisomers" or "stereoisomers" refer to compounds having one or more stereogenic centers that differ in chirality. Stereoisomers include enantiomers and diastereomers. As used herein, "tautomer" refers to alternative forms of compounds that differ in proton position, such as enol-ketone and imine-enamine tautomers. As used herein, a "salt" of a compound refers to an ion of the compound associated with a counterion ion. Salts of the compounds may be formed by neutralization of acids and bases. Salts may be derived from a variety of organic and inorganic counterions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium and tetraalkylammonium; and when the molecule contains basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate and oxalate. While the structures of the compounds disclosed herein may be shown in only one resonant form, it is understood that all resonant forms are included.

The dyes of the present disclosure may be prepared by any suitable method. For example, dyes may be prepared according to the methods shown in schemes I and II below.

In another aspect, provided herein are fluorescent dye-labeled polynucleotides prepared by automated oligonucleotide synthesis using the fluorescent dye phosphoramidites disclosed herein. The polynucleotide comprises a moiety derived from a compound of the present disclosure. As used herein, "fluorescent dye-labeled polynucleotide" or "labeled polynucleotide" refers to a polynucleotide prepared from a compound of formula (la) disclosed herein by automated oligonucleotide synthesis.

The labeled polynucleotides disclosed herein may comprise one or more additional moieties. In some embodiments of the disclosure, the labeled polynucleotide comprises a minor groove binder. In some embodiments, the labeled polynucleotide comprises an intercalator. In some embodiments, the labeled polynucleotide comprises a second fluorophore and/or a fluorescence quencher.

In general, polynucleotides labeled with the dyes disclosed herein are polynucleotides in which the backbone comprises 2' -deoxyribose or ribose. However, the labeled polynucleotide may comprise one or more modifications. In some embodiments, the polynucleotide comprises a sugar modification, e.g., a modified sugar. Various sugar modifications are useful. Some non-limiting sugar modifications include arabinose, d-arabinohexatol, 2-fluoroarabinose, xylulose, hexose or bicyclic sugar.

The labeled polynucleotides of the present disclosure may comprise one or more backbone modifications. In some embodiments, the polynucleotide comprises a backbone modification. In some embodiments, the backbone modification is selected from the group consisting of a modified sugar phosphate backbone, a locked nucleic acid backbone, a peptide backbone, a phosphotriester backbone, a phosphoramidate backbone, a siloxane backbone, a carboxymethyl ester backbone, an acetamidate (acetamidate) backbone, a carbamate backbone, a thioether backbone, a bridged methylene phosphonate backbone, a phosphorothioate backbone, a methylphosphonate backbone, an alkylphosphonate backbone, a phosphate backbone, an alkylphosphonate backbone, a dithiophosphate backbone, a carbonate backbone, a phosphotriester backbone, a carboxymethyl ester backbone, a methylthiophosphate backbone, a dithiophosphate backbone, a backbone having a para-ethoxybond, and a combination of any two or more of the foregoing. In a specific embodiment of the present disclosure, the backbone modification is a modified sugar phosphate backbone.

The labeled polynucleotides disclosed herein may comprise one or more modified or unnatural bases. Modified bases include modified thymine and cytosine bases (e.g., those disclosed in U.S. Pat. Nos. 9,598,455 and 9,598,456), 2, 6-diaminopurine bases, universal bases, and the like. The labeled polynucleotides disclosed herein can comprise non-nucleoside segments or non-nucleoside monomers (e.g., linkers such as poly (ethylene glycol) linkers).

In some embodiments, the polynucleotides disclosed herein are probes, e.g., 5' -nuclease PCR probes. In certain embodiments, the polynucleotide further comprises one or more additional labels, such as a fluorescence quencher. As will be appreciated by one of ordinary skill in the art, the position of the label within the oligonucleotide may vary and is not limited to the disclosure herein.

In some embodiments, provided herein are modified polynucleotides comprising a dye moiety as a fluorophore at one end of their sequence and a fluorescence quencher at the other end of their sequence, such that the fluorescence quencher inhibits the fluorescent signal of the fluorophore in the intact probe (i.e., an oligonucleotide used as a probe) via an energy transfer mechanism such as fluorescence resonance energy transfer ("FRET"). When the polymerase extends the primer along the template to which the probe also hybridizes, the 5' -nuclease activity of the polymerase cleaves the probe, thereby diffusing the fluorophore away from the fluorescence quencher, such that a fluorescent signal is now detected. The signal increases with each PCR cycle in proportion to the amount of probe that is cleaved and thus in proportion to the amount of amplification product (e.g., amplicon, target sequence). This allows direct detection and quantification of the target DNA sequence.

In some embodiments, the dye moiety is at least one nucleotide position from the end of the labeled polynucleotide sequence and the fluorescence quencher is linked to a base at least one nucleotide position from the other end of the modified polynucleotide. In some embodiments, the dye moiety and the fluorescence quencher are located within the probe. As will be appreciated by one of ordinary skill in the art, the location of the fluorophore and/or fluorescence quencher within the probe may vary and is not limited.

In some embodiments, the dye moiety and the fluorescence quencher are not at the terminus of the FRET probe. In some embodiments, the emission spectrum of the dye substantially overlaps the absorption spectrum of the fluorescence quencher. However, when quenching involves a collision mechanism, or overlap increases, for example due to reaction conditions or probe structure, such spectral overlap is less important or not required.

There are a number of practical guidelines in the art for selecting an appropriate fluorophore-quencher for a particular probe. See, e.g., FLUORESCENCE SPECTROSCOPY (Marcel Dekker, new York, 1971). Quenchers useful in the probes disclosed herein include bis-azo quenchers (e.g., those disclosed in U.S. patent No. 6,790,945), quenchers obtainable from Biosearch Technologies, inc (Black Hole) ^TM Quenching agent: BHQ-1, BHQ-2, and BHQ-3), TAMRA, carboxytetramethyl rhodamine, 4- ((4- (dimethylamino) phenyl) azo) benzoic acid (Dabcyl),

Quencher, & gt>

Quenching agent, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ) -I and 2- [6- (1, 3-dihydro-2H-isoindol-2-yl) -9- {2- [ (4- [ (2, 5-dioxopyrrolidin-1-yl) oxy)]Carbonyl piperidin-1-yl) sulfonyl]Phenyl } -3H-xanthen-3-subunit]-2, 3-dihydro-1H-isoindolium chloride (2- [6- (1, 3-dihydro-2H-isoinondol-2-y 1) -9- {2- [ (4- [ (2, 5-dioxaborolidin-1-y 1) o-xy)]carbonyl piperidin-1-y1)sulfo-nyl]phenyl}-3H-xanthen-3-ylidene]-2，3-dihydro-1H-isoin dolium chloride) (QSY 21) and other quenchers known in the art.

In yet another aspect, disclosed herein is a method for preparing a labeled conjugate of a ligand comprising contacting the ligand with a compound of the formula provided herein in a suitable solvent under conditions sufficient to covalently attach the compound to the ligand, thereby forming a dye-labeled conjugate. Suitable ligands include biomolecules (e.g., polynucleotides, oligonucleotides, proteins, antibodies, peptides or polysaccharides), synthetic polymers (e.g., polymers having an olefinic backbone, such as polyacrylic acid), and solid supports (e.g., controlled pore glass or polystyrene).

In some embodiments, the ligand is a polynucleotide. In some embodiments, the conditions sufficient to covalently attach a compound of the present disclosure to a ligand, i.e., an oligonucleotide or polynucleotide, are automated phosphoramidite oligonucleotide synthesis conditions. Automated phosphoramidite oligonucleotide synthesis conditions for synthesizing and deprotecting synthetic oligonucleotides are well known in the art and are described, for example, in current protocols in nucleic acid chemistry (Current Protocols in Nucleic Acid Chemistry), volume I, beaucage et al, editions, john Wiley & Sons,2002, the disclosure of which is incorporated herein by reference.

The phosphoramidite method of oligonucleotide synthesis, such as DNA, is considered the standard synthesis method used in most automated synthesizers. The building blocks used for synthesis are often referred to as nucleotide building blocks, monomers or nucleoside phosphoramidites, which are activated nucleoside derivatives (phosphoramidites). An acid cleavable protecting group, typically a Dimethoxytrityl (DMT) group, is used to protect the 5 '-end of the nucleoside and a beta-cyanoethyl group is used to protect the 3' -phosphite moiety. The monomer may also include additional groups for protecting other moieties such as reactive primary amines in the nucleobase. The protecting groups are selected to prevent branching or other unwanted side reactions during the synthesis. The skilled artisan will be able to readily select protecting groups having properties suitable for use under specific synthesis and deprotection and/or cleavage conditions. For example, various amine protecting groups are taught in Greene & Wuts, "protecting groups in organic chemistry (Protective Groups In Organic Chemistry)," 3 rd edition, john Wiley & Sons,1999 (hereinafter "Green & Wuts").

Typically, oligonucleotides are synthesized on solid supports such as Controlled Pore Glass (CPG) or polystyrene packed columns, membranes or similar materials. Oligonucleotides are typically synthesized from the 3 'end to the 5' end. The first nucleotide building block or monomer is typically immobilized to the support via a linker such as long chain alkylamine controlled pore glass (LCAA-CPG).

In some embodiments, the synthetic method employing phosphoramidite reagent involves multiple rounds of: (i) DMT deprotection to reveal free hydroxyl groups can be achieved, for example, by treatment with 2.5% or 3% dichloroacetic acid or trichloroacetic acid in dichloromethane; (ii) Coupling of nucleosides or other phosphoramidite reagents to free hydroxyl groups, which can be performed, for example, in acetonitrile containing tetrazoles (e.g., 0.45M or 0.5M tetrazoles); (iii) Oxidation, which can be effected, for example, by using I ₂ 2, 6-lutidine/H ₂ O treatment; and capping, which can be performed, for example, by treatment with 6.5% acetic anhydride in Tetrahydrofuran (THF), activation with 10% 1-methylimidazole (NMI) in THF.

Other conditions for performing the various steps in the synthesis are also known in the art and may be used herein. For example, phosphoramidite coupling may be performed in acetonitrile containing 0.25M 5-ethylthio-1H-tetrazole, 0.25M 4, 5-Dicyanoimidazole (DCI), or 0.25M 5-benzylthio-1H-tetrazole (BTT). It is possible to use 0.1M, 0.05M or 0.02M in THF/H ₂ I in O/pyridine (7:2:1) ₂ Oxidation is performed. The capping can be performed by treatment with THF/lutidine/acetic anhydride, followed by treatment with 16% NMI in THF.

Removal of any protecting groups and cleavage from the synthesis reagents is typically achieved by treatment with concentrated ammonium hydroxide at 60 ℃ for 1-12 hours, although nucleoside phosphoramidites protected with groups that can be removed under milder conditions such as by treatment with concentrated ammonium hydroxide at room temperature for 4-17 hours or with 0.05M potassium carbonate in methanol or with 25% t-butylamine in water/ethanol are also known and may be used.

The term "cleavage" in reference to solid phase oligonucleotide synthesis refers to breaking the bond linking the oligonucleotide to the solid support. In some embodiments, cleavage involves hydrolysis of the succinate linkage between the 3' hydroxyl group of the ligated oligonucleotide and the solid support.

The term "deprotection" as used herein refers to the removal of a protecting group from the exocyclic amine of a heterocyclic base of an oligonucleotide. In general, deprotection involves the hydrolysis of an amide moiety consisting of an exocyclic amine and an amino protecting group such as benzoyl or isobutyryl. Various deprotection techniques and methods are known in the art.

In another aspect, provided herein are kits comprising labeled polynucleotides or compounds of the disclosure. In some embodiments, the kit is a PCR diagnostic kit comprising one or more polynucleotides of the present disclosure. In some embodiments, the kit is an automated molecular diagnostic kit.

Although each element of the present invention is described herein as comprising a plurality of embodiments, it should be understood that each embodiment of a given element of the present invention can be used with each embodiment of the other elements of the present invention unless otherwise indicated, and each such use is intended to form a different embodiment of the present invention.

References, patent applications, and scientific literature cited herein are incorporated by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Also, any conflict between a definition of a word or phrase as understood in the art and a definition of a word or phrase specifically taught in this specification shall be resolved in favor of the latter.

The invention is illustrated by the following examples. These examples are included for illustrative purposes only and are not intended to limit the invention.

Examples

Protons [ ] ¹ H,400 MHz) and phosphorus% ³¹ P,160 MHz) Nuclear Magnetic Resonance (NMR) spectra were obtained on a Bruker Biospin 400 instrument. NMR samples in DMSO-d ₆ And CD (compact disc) ₃ CN was prepared and the residual protonated solvent was used as an internal chemical shift standard. LCMS data were obtained by electrospray ionization (ESI) on Agilent 1200 series (LC/MSD Trap XCT Plus) and Agilent 1260 index (6130 quadrupole LC/MS) instruments. Using Biotage Isolera LS and Teledyne ISCO

The Combi Flash instrument performs an automatic chromatography on silica gel 60. Analytical thin layer chromatography was performed on aluminum back silica gel 60F254 and plates were visualized under uv lamps (254 and 365 nm). Unless otherwise indicated, all reagents were from commercial sources.

Polynucleotides comprising compounds of the present disclosure were synthesized in DMT removal-coupling-blocking-oxidation-blocking cycles using a standard 200nmol DNA protocol on a MerMade-12 oligonucleotide synthesizer. The coupling time of the dye phosphoramidites of the present disclosure can be adjusted accordingly.

For polynucleotides containing 5' dyes, synthesis is completed after the last dye phosphoramidite coupling cycle. In the case of dye phosphoramidite internal incorporation, more polynucleoside monomer is added after dye coupling. The last DMT group remains on the polynucleotide.

The fully assembled polynucleotide was cleaved from the solid support and deprotected by 30% ammonium hydroxide at 55℃for 10-12 hours. After removal of ammonia, the oligonucleotides were analyzed and purified by reverse phase HPLC (RP-HPLC) on a C18Gemini column eluting with a linear gradient of pH 7 acetonitrile/0.1M triethylammonium bicarbonate. After removal of the DMT groups, the polynucleotide was purified a second time. Excitation and emission spectra of the labeled oligonucleotides were recorded on an Agilent Cary fluorometer (200 nM oligonucleotides, 0.1M Tris buffer, pH 8).

An exemplary synthetic method of an exemplary fluorescent dye phosphoramidite of the present disclosure (e.g., compound D) is shown in reaction scheme I.

First, starting alcohol compound a (e.g., a compound of table I) is acylated in situ with bis (4-nitrophenyl) carbonate in the presence of a base, followed by nucleophilic substitution of the 4-nitrophenyl ester with a secondary amine to form a urethane linkage to yield intermediate C. The product may be isolated by flash chromatography. Compound C was converted to phosphoramidite D using standard phosphoramidite D under anhydrous conditions to remove acid. The reaction mixture and products are protected from light and air (oxygen). The product was purified using silica gel flash chromatography.

Reaction scheme I.

Alcohol compound a, such as the compound of table 1, can be prepared according to the method shown in reaction scheme II.

Reaction scheme II.

Table 1.

Preparation of precursor alcohol A (Compound 19A).

2, 3-dihydro-2, 3-trimethyl-1- (2-hydroxyethyl) -indol-5-ol (prepared according to the procedure of WO 2019/2174470; 28.0 mmol) and 4-fluororesorcinol (56.0 mmol) are suspended in 50mL of methanesulfonic acid under a dry, inert atmosphere. The solution was heated at 55 ℃ until it became homogeneous. 2,4, 5-trimethylbenzaldehyde (61.6 mmol) in methanesulfonic acid (15 mL) was added to the solution, and the mixture was heated at 55deg.C for 1.5 hours. The reaction flask was placed inTo the ice bath, 150g crushed ice and 125ml water were added. The mixture was treated with THF/CHCl ₃ (1:2) extraction three times and combining the organic layers with saturated NaHCO ₃ Extracted and subjected to anhydrous Na ₂ SO ₄ And (5) drying. Silica gel chromatography using acetonitrile/water provided 3.67mmol (13%) of dye compound 19A with excitation/emission maxima of 528nm/546nm, respectively.

Preparation of exemplary phosphoramidites (Compound 19D).

Triethylamine was added to a solution of compound 19A in anhydrous N, N-dimethylformamide under an argon atmosphere, followed by one-time addition of BNPC. The reaction was incubated for 40 minutes. Linker compound L1 was added to the reaction mixture and the reaction mixture was incubated for 2.5h at ambient temperature. The reaction mixture was transferred to a separatory funnel, and the reaction flask was rinsed with ethyl acetate. The solution was extracted with water. The aqueous layer was discarded, and the organic layer was further washed with deionized water and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate with stirring for 18h. The crude product was purified by flash chromatography to give compound 19C.

Compound 19C was dissolved in anhydrous dichloromethane under argon and anhydrous N, N-diisopropylethylamine was added via syringe with stirring. The reaction flask was placed in an ice bath for 10 minutes to cool the reaction mixture to < 10 ℃. Pam-Cl was added dropwise via syringe over 1 min and the reaction mixture was removed from the ice. The reaction mixture was protected from light by wrapping the flask with aluminum foil and allowed to gradually warm to room temperature for 2.5 hours. The reaction mixture was diluted with ethyl acetate and transferred to a separatory funnel under argon. After transfer, the reaction mixture was extracted with 10% saturated sodium bicarbonate solution. The aqueous layer was discarded and the organic layer was extracted with saturated sodium chloride to remove water. The organic layer was dried over anhydrous sodium sulfate under argon, protected from light. After drying, the solution was filtered and the solvent was evaporated to give a red foamy crude product. The crude product was purified by flash chromatography. The product compound 19D was analyzed by HPLC, LC-MS and NMR.

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims (32)

1. A compound of formula I or a stereoisomer, salt or tautomer thereof:

Wherein:

x is H, halogen or C ₁ -C ₅ An alkyl group;

R ¹ 、R ² 、R ³ and R is ⁴ Independently H or optionally substituted C ₁ -C ₆ An alkyl group;

R ⁵ 、R ⁶ 、R ⁷ 、R ⁸ and R is ⁹ Independently H, halogen or optionally substituted C ₁ -C ₆ An alkyl group;

L ¹ is optionally substituted C ₂ -C ₁₀ Alkylene or optionally substituted C ₂ -C ₅₀ A heteroalkylene group;

L ² and L ³ Independently optionally substituted C ₂ -C ₁₀ Alkylene or optionally substituted C ₂ -C ₃₀ A heteroalkylene group;

Q ¹ is a hydroxyl protecting group;

z is CH, N, NHC (O) N or OC (O) N;

y is OH, OP (OCH) ₂ CH ₂ CN)NR ¹⁰ R ¹¹ Or a solid support; and

R ¹⁰ and R is ¹¹ Independently optionally substituted C ₁ -C ₆ An alkyl group.

2. The compound of claim 1, wherein X is C1, br, or F.

3. The compound of claim 1, wherein X is Cl.

4. A compound according to any one of claims 1 to 3 wherein the solid support is a controlled pore glass or polystyrene.

5. A compound according to any one of claims 1 to 3, wherein the compound is a compound of formula (IA) or a stereoisomer, salt or tautomer thereof:

6. the compound of any one of the preceding claims, wherein R ¹ Is H.

7. The compound of any one of the preceding claims, wherein R ² Is methyl.

8. The compound of any one of the preceding claims, wherein R ³ Is methyl.

9. The compound of any one of the preceding claims, wherein R ⁴ Is methyl.

10. The compound of claim 1, wherein the compound is a compound of formula (IB):

11. the compound of any one of the preceding claims, wherein L ¹ Is C ₂ -C ₆ Alkylene or- (CH) ₂ CH ₂ O) _m CH ₂ CH ₂ -wherein m is an integer from 1 to 10.

12. The compound of any one of the preceding claims, wherein L ¹ Is C ₂ An alkylene group.

13. The compound of any one of the preceding claims, wherein Z is OC (O) N.

14. The compound of any one of the preceding claims, wherein the compound is a compound of formula (IC) or a stereoisomer, salt, or tautomer thereof:

wherein m is an integer of 1 to 10.

15. The compound of claim 1, wherein the compound is a compound of (ID) or a stereoisomer, salt, or tautomer thereof:

wherein W is NH or O, and L ⁴ Is optionally substituted C ₂ -C ₁₀ Alkylene or optionally substituted C ₂ -C ₅₀ A heteroalkylene group.

16. The compound of any one of the preceding claims, wherein Q ¹ Is trimethylsilyl, TBDMS, acetyl, dimethoxytrityl or trityl.

17. The compound of any one of the preceding claims, wherein R ¹⁰ And R is ¹¹ Is isopropyl.

18. The preceding claimThe compound of any one of claims, wherein R ⁵ Is methyl.

19. The compound of any one of the preceding claims, wherein R ⁷ Is methyl.

20. The compound of any one of the preceding claims, wherein R ⁸ Is methyl.

21. The compound of any one of the preceding claims, wherein R ⁶ And R is ⁹ Is H.

22. The compound of claim 1, wherein the compound is a compound of formula (IE) or a stereoisomer, salt, or tautomer thereof:

23. a labeled polynucleotide prepared using an automated phosphoramidite synthesis comprising a residue of the compound of any one of claims 1-22.

24. The labeled polynucleotide of claim 23, wherein the labeled polynucleotide further comprises a fluorescence quencher.

25. The labeled polynucleotide of claim 23, wherein the labeled polynucleotide is attached to a solid support.

26. The labeled polynucleotide of claim 25, wherein the solid support is a controlled pore glass bead, a polystyrene bead, a magnetic bead, or a microplate.

27. A method for preparing a labeled conjugate of a ligand comprising contacting in a suitable solvent, in an amount sufficient to drive Contacting the ligand with the compound of any one of claims 1-22 under conditions in which the compound is covalently attached to the ligand, thereby forming a labeled conjugate; wherein Y is OP (OCH) ₂ CH ₂ CN)NR ¹⁰ R ¹¹ And R is ¹⁰ And R is ¹¹ Independently optionally substituted C ₁ -C ₆ An alkyl group.

28. The method of claim 27, wherein the ligand is a polynucleotide or a solid support.

29. The method of claim 27, wherein the ligand is a polynucleotide.

30. The method of claim 27, wherein the conditions sufficient to covalently attach the compound to the ligand are automated phosphoramidite oligonucleotide synthesis conditions.

31. A kit comprising the labeled polynucleotide of claim 23 or claim 24.

32. The kit of claim 31, wherein the kit is a PCR diagnostic kit.